544 G. C. WOOD I958The present work shows that a soluble degrada-

tion product of elastin can be converted into aninsoluble form, having many of the characteristicproperties of the original elastin. The way in whichelastin is formed in vivo is not known with cer-tainty but it is at least a possibility that it isformed from a soluble precursor by a mechanismsimilar to reconstitution.

SUMMARY

1. The high-molecular-weight a-fraction ofsoluble elastin prepared from ox ligamentumnuchae (Adair et al. 1951) becomes insoluble onprolonged heating of aqueous solutions undercertain conditions.

2. Heat-precipitated elastin has many of thephysical properties of purified elastin and it isconcluded that the protein molecules have essenti-ally the same configuration in the two materials.

3. Differences between heat-precipitated (re-constituted) elastin and purified elastin may beattributed to degradation of the latter when it isconverted into soluble elastin.

4. The precipitation of elastin appears to con-sist of two processes, namely a rapid one whichmay involve configurational changes and results incoacervation and a slow one which is probably dueto aggregation of the protein molecules.

5. The bearing of the results on the structure ofelastin and its fibrogenesis is discussed.

I wish to thank Miss H. Saxl for studying the histology ofpurified and precipitated elastin and Dr W. G. Horton forexamining precipitated elastin by X-ray diffraction. I alsowish to thank Miss H. Saxl, Dr D. A. Hall and ProfessorR. E. Tunbridge for helpful discussion and criticism andMiss J. Waddington for technical assistance.

REFERENCES

Adair, G. S., Davis, H. F. & Partridge, S. M. (1951).Nature, Lond., 167, 605.

Astbury, W. T. (1940). J. Soc. Leath. Tr. Chem. 24, 69.Balo, J. & Banga, I. (1950). Biochem. J. 46, 384.Bowen, T. J. (1953). Biochem. J. 55, 706.Burton, A. C. (1954). Physiol. Rev. 34, 619.Flory, P. J. (1956). J. Amer. chem. Soc. 78, 5222.Hall, D. A. (1955). Biochem. J. 59, 459.Hall, D. A. (1957a). Arch. Biochem. Biophys. 67, 366.Hall, D. A. (1957b). Connective Ti8we, C.1.O.M.S. Symp.,

p. 238. Ed. by Tunbridge, R. E., Keech, M. K., Dela-frcsnaye, J. F. & Wood, G. C. Oxford: BlackwellScientific Publications.

Hall, D. A., Reed, R. & Tunbridge, R. E. (1952). Nature,Lond., 170, 264.

Hall, D. A., Reed, R. & Tunbridge, R. E. (1954). Exp. CellRes. 8, 35.

Lansing, A. I., Rosenthal, T. B., Alex, M. & Dempsey,E. W. (1952). Anat. Rec. 114, 555.

Lloyd, D. J. & Garrod, M. (1946). Fibrous Protein8, Soc. ofDyers and Colourists Symp., p. 24.

Meyer, K. H. & Fern, C. (1936). Arch. Physiol. 238, 78.Partridge, S. M. & Davis, H. F. (1955). Biochem. J. 61,

21.Partridge, S. M., Davis, H. F. & Adair, G. S. (1955).

Biochem. J. 61, 11.Ramachandran, G. N. & Santhanam, M. S. (1957). Proc.

Ind. Acad. Sci. A, 45, 124.Saxl, H. (1957). Gerontologia, 1, 142.Seifter, S., Dayton, S., Norrie, B. & Muntwyler, E. (1950).

Arch. Biochem. Biophy8. 25, 191.Treloar, L. R. G. (1949). Physics of Rubber Elasticity,

p. 19. Oxford: Clarendon Press.Waugh, D. F. (1954). Advanc. Protein Chem. 9, 342.W6hlisch, E., Weitnauer, H., Gruning, W. & Rohrback, R.

(1943). Kolloidzschr. 104, 14.Wood, G. C. (1954). Biochim. biophys. Acta, 15, 311.Woods, H. J. (1946). J. Colloid Sci. 1, 407.

The Site of Protein Synthesis in Bacillus megaterium

BY J. A. V. BUTLER, A. R. CRATHORN AND G. D. HUNTERChe8ter Beatty Re8earch Institute, In8titute of Cancer Re8earch: Royal Cancer Hospital,

Fulham Road, London, S.W. 3

(Received 18 November 1957)

In recent years much work has been done on theincorporation of isotopically labelled amino acidsinto the various fractions of mammalian cells. Ithas been found that the ribonucleoprotein particlesassociated with the microsomal fraction becomelabelled at a very early stage (Borsook, Deasy,Haagen-Smit, Keighley & Lowy, 1950; Keller,Zamecnik & Loftfield, 1954; Littlefield, Keller,Gross & Zamecnik, 1955; Simkin & Work, 1957;

Cohn & Butler, 1957), although evidence for a pre-liminary activation process in the 'soluble fraction'has been given (Hoagland, Keller & Zamecnik,1956; Hoagland, Zamecnik & Stephenson, 1957).On the other hand, little information has beenobtained about the sites of protein synthesis in thebacterial cell. It has, however, been shown that inTorulop8i8 Uili8 absorbed amino acids do not existin the free state at all but are bound in such a way

SITE OF PROTEIN SYNTHESIS IN B. MEGATERIUM5that they are not freely exchangeable with aminoacids in the surrounding medium (Cowie & Walton,1956), and Gale & Folkes (1955) found that a cell-envelope fraction from Staphylococcus aureus wascapable of developing enzyme activities and of in-corporating labelled amino acids into protein.These two reports indicated that the cell wall orcytoplasmic membrane might play an importantpart in the synthesis ofprotein by micro-organisms.

Bacillus megaterium seemed to us a suitablemicro-organism for studies of the site of proteinsynthesis in view of the work of Weibull (1953a,1955). He had shown that it is easily possible toremove the cell wall by treatment with lysozyme,leaving, when suitable precautions are taken, theprotoplasm of the cell in the form of sphericalprotoplasts.

It has been shown (McQuillen, 1956) that theseprotoplasts are able to synthesize proteins, nucleicacids and enzymes, including adaptive enzymes;they can also grow and divide and allow the de-velopment of endospores. When shaken violently,or placed in media of low osmotic pressure, theycdn be lysed, with the resultant liberation into themedium of the soluble cell constituents. Twostructures then remain: empty vesicles or 'ghosts'derived from the cytoplasmic membrane thatoriginally surrounded the protoplasts and smallgranules. The membranes and granules may beisolated by differential centrifuging (Weibull,1953 b), and the separated granules appear toconsist largely of polymerized ,-hydroxybutyricacid. If the membrane-granule fraction is sedi-mented under the conditions described in thispaper, the clear supematant fraction (cytoplasm)deposits little further sediment when centrifugedfor 30 min. at 100 000 g, so that there appears tobe no close equivalent of the mammalian micro-some fraction present (Hunter, Crathorn & Butler,1957).In the experiments reported here a study has

been made of the incorporation of labelled aminoacids into the cell wall, the cytoplasmic membraneand the supematant fractions. The incorporationhas been studied in (1) the intact bacterium;(2) intact and broken protoplasts; (3) the membraneand supematant fractions of the protoplasts. Apreliminary account of part of this work has beengiven (Crathom & Hunter, 1958).

METHODS

OrganisM. We are indebted to Dr K. McQuillen forsupplying the original strain KM of Bacillus megaterium.It has been maintained by frequent subculture on peptone-agar slopes. By progressive selection, a strain has beenisolated that forms very stable protoplasts when treatedwith lysozyme. These can be treated almost as whole cells

35

when suspended in neutral media containing 0-5M-phosphate and can then be disintegrated only after veryviolent shaking.The organism was usually grown at 30° in a glucose-

salts solution (McQuillen, 1955) medium, although on someoccasions the glucose was replaced by 0-5% (w/v) peptone(British Drug Houses Ltd., Poole, England) withoutapparently affecting any of the biochemical propertiesstudied here. Large batches for experimental purposes wereprepared in aerated Roux bottles or 51. shake flasks.

Experiments with whole cells. Whole cells from largebatches grown overnight were harvested and washed, sus-pension densities being determined from the absorption at420 mFi on a Unicam spectrophotometer (model SP. 600)previously calibrated against the organism. For tracerexperiments the cells were resuspended in the glucose-salts medium and re-incubated aerobically at 30° for 1 hr.before adding any [14C]amino acid. After further aerobicincubation for the required length of time, the cells weresedimented and resuspended in a medium containing 0-5M-sodium citrate, 0-5% (w/v) of casein digest (Bacto-Casamino acids; Difco Laboratories Inc., Detroit, Mich.,U.S.A.), and 0-02% of lysozyme. Protoplasts were col-lected by centrifuging after standing overnight at 2°, andthe digested cell-wall material was obtained in the super-natant medium. The protoplasts were finally lysed bypouring into 4 vol. of water and the fraction containing thecytoplasmic membrane and granules was collected bysedimentation at 10 000 g for 10 min. In some experimentsit was found necessary to sediment at 20 000g for 20 min.to obtain a firm pellet.The supernatant fraction from this last centrifuging

contained the cytoplasm of the cell diluted with the sus-pension medium. It was found that estimations of theradioactivity of protein in this fraction were in many caseslittle affected if the cell-wall digest and free amino acidswere also present, and in some experiments a better controlof timing was attained by pouring the original cell sus-pension directly on to ice at the end of the period ofincubation with the labelled amino acid. Lysozyme wasadded to a final concentration of0-02% and the cytoplasmicmembranes were isolated as before after standing overnightat 20.

In some cases the cytoplasmic-membrane fractions werefurther treated with ethylenediaminetetra-acetic acid(EDTA) (1% sodium salt; pH 7) at 300 for 1 hr. This hadthe effect of solubilizing many of the granules and bringinginto solution about one-third of the protein and some of thedeoxyribonucleic acid (DNA). The actual amount of DNAsolubilized in this way was variable (35-65 %). Vennes &Gerhardt (1956) claim that it is possible to remove DNAcompletely from the cytoplasmic membranes of B. mega-terium by treating protoplasts with a mixture of EDTAand deoxyribonuclease, but under conditions where theactivity of the enzyme was probably very small.

Fxperiments with protoplasts. Protoplasts were preparedby gently shaking suspensions of whole cells at a density of1 mg./ml. (dry wt.) in air at 30° in a medium containing0-5M-sodium citrate, 0-5% of casein digest and 0-02% oflysozyme. The complete conversion into protoplastsusually took about half an hour under these conditions,sometimes much longer. -On occasion, suspensions wereobtained that were partially or completely insensitive to theaction of lysozyme under these conditions. We can offer no

Bioch. 1958, 69

Vol. 69 545,

J. A. V. BUTLER, A. R. CRATHORN AND G. D. HUNTERexplanation of this phenomenon; fortunately it is not ofvery frequent occurrence. The protoplasts were sedimentedand resuspended for incorporation experiments in 'C'solution (McQuillen, 1955) containing, in addition, 0-5m-KH,P04 and 1% of glucose, the whole brought to pH 7with sodium hydroxide. In this medium they were verystable and could be shaken vigorously enough to maintainadequate aeration without appreciable lysis taking place.It is always advisable, however, to follow closely by micro-scopic examination, any metabolic experiments with wholeprotoplasts.

After shaking at 300 in this medium for 1 hr. labelledamino acids were added and any further period of incuba-tion of the protoplasts could be defined clearly by pouringthe whole or part of the suspension directly on to 4 vol. ofice-water, when instantaneous lysis occurred. The cyto-plasmic membrane fraction was then separated at oncefrom the cytoplasmic fraction by centrifuging at 10 000-20 000g for 10-20 mi.

Experiments with broken protoplasts and isolated mem-branes. Thick suspensions of protoplasts (20 mg./ml.) inphosphate-'C'-glucose medium, detailed in the previoussection, were broken by rapid oscillation in a Mickle tissuedisintegrator. The suspension was then diluted 20-foldwith the same medium and incubated aerobically at 300 for30 min. 14C-Labelled amino acid was then added in orderto study the incorporation of the amino acid into proteinand the incubation continued for varying times, eventuallybeing stopped by rapid cooling. The membrane fractionwas then separated from the cytoplasm and incubationmedium by sedimentation at 20 000g for 20 mi.

In other experiments, the cytoplasmic membranes wereseparated from the cytoplasm by centrifuging and theincorporation of [14C]amino acid was studied in the twoseparated fractions. The protoplasts could also be brokenby diluting twice with water and shaking vigorously byhand for 2 min., and the properties of the isolated mem-branes and cytoplasm thus obtained were very similar tothose of the corresponding fractions obtained by theprevious treatment. For incorporation studies on theisolated membranes, they were usually resuspended in theinorganic 'C' solution with 1% of glucose added.When transfer experiments were carried out with cyto-

plasmic membranes labelled in vitro, the labelling mediumwas removed and the washed membranes were then re-incubated aerobically with non-labelled cytoplasm at 30°for the appropriate period of time. The cytoplasm and themembranes were then separated once more by centrifugingat 20 000g for 20 mi.Attempts to obtain incorporation of [14C]amino acids

into the protein of isolated cytoplasm were made by incu-bating the cytoplasm aerobically at 30° in the presence ofvarious additives.

Estimation of nucleic acids. Ribonucleic acid (RNA) wasdetermined in whole cells and subcellular fractions by theorcinol method (Cerriotti, 1955). DNA was determined bythe indole method (Cerriotti, 1952). Samples of DNA andRNA which had been prepared from whole cells by adetergent method and separated by differential centri-fuging (Hunter & Butler, 1956) were used as standard.These separated nucleic acids also served as standards inestimating the proportions of RNA and DNA in mixednucleic acids extracted by treatment of various fractionswith sodium dodecyl sulphate. Base analyses of extracted

nucleic acids (Wyatt, 1951) were carried out after hydro-lysis with perchloric acid (Marshak & Vogel, 1951).

Preparation of samples for radioactivity assay. Proteinsamples for counting were obtained from suspensions orsolutions by adding aqueous trichloroacetic acid to a finalconcentration of 5 % (w/v), and then removing lipids andnucleic acids from the precipitated proteins as describedpreviously (Crathorn & Hunter, 1957). Where the totalradioactivity of a given fraction was required it wasevaporated to dryness at 900 and counted directly.

Assays of radioactivity. These were carried out wherepossible on 'infinitely thick' samples of protein or driedbacterial fractions plated on 1 cm.2 polythene disks(Popj'ak, 1950), an EHM2S end-window Geiger-Mullercounter in conjunction with an N529 scaling unit (manu-factured by Ekco Electronics Ltd., Southend-on-Sea,Essex) being used. The counter was housed in a lead castlemanufactured by E.R.D. Ltd., Slough, Bucks. Under theseconditions a 14C-labelled protein sample of specific activity1 jc/g. would give a counting rate of about 500 counts/min.when plated at 30 mg./cm.2 (i.e. an efficiency of 1-2 %).Samples of less than 'infinite' thickness were plated uni-formly in the disks, and the counts obtained corrected to'infinite' thickness by making use of an experimentallydetermined curve obtained with bacterial protein of knownspecific activity.

Materials. Generally labelled L-amino acids weresupplied by the Radiochemical Centre, Amersham, Bucks.[1-14C]Glycine (10-3 fc/mg.) and DL-[3-14C]phenylalanine(12-0pc/mg.) were synthesized in these Laboratories byDr P. Brookes and Dr V. C. E. Burnop. All the [L4C]aminoacids were used throughout this work without any dilutionwith unlabelled amino acid.

RESULTS

Experiments with whole cells. In order to deter-mine the extent to which the cell wall participatedin incorporation of amino acid, various 14C-labelledamino acids were added to whole cells of B.megateriurn growing in the full glucose-saltsmedium and incubations were continued for 5 or30 min. before removing the cell walls with lyso-zyme. The results are summarized in Table 1; itcan be seen that the specific activity of the cell-wall 'protein' is much lower than that of the proto-plast protein in all cases. Of course, this might nothave been true if any of the three amino acids mostabundant in the cell wall of B. megaterium (alanine,glutamic acid and diaminopimelic acid) had beenused. But it showed that the cell wall played nomajor part in the early stages of the synthesis ofthe bulk of the protein of the cell.The rate of incorporation of 14C-labelled amino

acids into the proteins of subcellular fractions ofthe protoplasts was next studied. A typical result,in which [1-14C]glycine was used, is shown in Fig. 1.For periods of incubation up to about 6 min. theprotein of the membrane fraction has the highestspecific radioactivity, but thereafter the cyto-plasmic proteins become more highly radioactive.

546 I958

SITE OF PROTEIN SYNTHESIS IN B. MEGATERIUM.

Table 1. Oomwparison of the incorporation of l4Caino acids into the proteins of ceU wall8 and protoplwats

Labelled- amino acids (2 c) were added to cells (100 mg. dry wt. at 1 mg./ml.) growing in a glucose-salts medium, andthe incubation was continued as before for 5 or 30 min. The cells were then sedimented, washed and the cell walls digestedwith lysozyme as described in the Methods section of this paper.

Amino acid addedL-['4C]LeucineL-[14C]Lysine[1-14C]GlycineDL-[3-L4C]Phenylalanine

Specific activity ofcell-wall protein

(Ic/g.) after

5 min. 30 min.0-09 0-240-37 0-390-35 1-650-14 1-3

Specific activity ofprotoplast protein

(Ac/g.) afterIr

A

5 min. 30 min.0-71 2-859-8 16-57*7 7-83.5 8-5

1.

-e 1-4

141-

021-

0.8

0 5 10 Is 20 25 30Time (min.)

Fig. 1. Incorporation of [1-14C]glycine into the protein ofsubcellular fractions ofwhole cells ofBacillus megaterium,incubated aerobically in an inorganic salts medium withglucose. The amino acid (10 pc) was added to 1 g. dry wt.of cells growing at 1 mg./ml. in this medium. Sampleswere taken at various times and analysed as described inthe Methods section. 0, Cytoplasm; 0, cytoplasmicmembrane; A, EDTA digest.

The proteins of the EDTA digests had, in general,a lower specific radioactivity than either of theother fractions, except at the very shortest periodsof incubation when they had slightly higher specificradioactivities than the cytoplaanmic proteins.Similar results were obtained with other labelledamino acids, and it was usually unnecessary toseparate cell-wall protein (or polypeptide material)from the cytoplasmic proteins, as most of it was

u 1-2 -

E 1-0 \

s0 X~~~>_ 0.8 _

0 0-60

0-4 -

0-2

0 5 10 15 20Time (min.)

Fig. 2. Incorporation of L-[14C]lysne into subcellularfractions of whole cells of BaeiUs megaterium, incubatedaerobically in an inorganic salts medium with glucose.The amino acid (12 ,tc) was added to 2 g. (dry'wt.) of cellsgrowing at 1 mg./ml. in the medium. Samples (333-3 ml.each) were taken at various times and analysed asdescribed in the text. 0, Cell wall; 0, cytoplasm; *,non-protein from cytoplasm; A, EDTA digest; 0, cyto-plasmic membrane.

removed during the precipitation and washingprocedures, and it did not greatly affect theobserved specific activity of the cytoplasmicprotein.However, in some experiments the curves for the

specific radioactivities of the different fractionswere closer than in others, particularly if thelysozyme-digestion procedure was performed at ahigher temperature than 2° or for a prolongedperiod. It seemed likely. that an unknown amountof equilibration of radioactive protein between the

35-2

Vol. 69 547

J. A. V. BUTLER, A. R. CRATHORN AND G. D. HUNTERvarious fractions was taking place as long as thecells remained intact.At the same time the rate of incorporation of

L-[14C]lysine into the various cellular fractions ofthe organism was studied as before, but the*fractions were dried at 80° and the total activitypresent in each fraction, including the non-proteinmaterial, was determined. The results are shown inFig. 2. A very large proportion of the added amino

9

8

7

-I?

(44#u,

2

0

I958

acid was fixed in the cell wall and the bulk of theremainder was found in the cytoplasmic fraction.Two further points of interest were: first, theamount of label in the membrane and EDTAdigests could at all times be accounted for almostentirely by the labelled protein present and,secondly, that the total radioactivity of the non-protein material present in the cytoplasm remainedconstant at all times of incubation from 30 sec. to

(a)

Time (min.)

-

uI-,

t!

4,u

._U)

15

(c)

0 2 4 6 8 10Time (min.) Time (min.)

Fig. 3. Incorporation of [O4C]amino acids into the proteins of the -membrane and cytoplasm after incubation ofprotoplasts of BaciUus megaterium in a medium oontaining 0-5M-KH2PO4, other inorganic salts and glucose.In each case lOJhc of labelled amino acid was added to protoplasts from 1-15 g. (dry wt.) of whole cells growingat 1 mg./ml., and samples were taken and analysed as described in the Methods section. (a) Incorporation of[l-14C]glycine; (b) incorporation of L-[14C]lysine; (c) incorporation of L-[14C]vahne. 0, Cytoplasm; 0, cytoplasmicmembrane.

548

-O.:

4._bO

.4u

I

SITE OF PROTEIN SYNTHESIS IN B. MEGATERIUM

20 min. These last figures were obtained bydifference; after assay of the total activity, thedried cytoplasm was extracted with cold trichloro-acetic acid and the radioactivity of the protein inthe residue determined after the removal of lipidand nucleic acid material by standard methods.The enormous reservoir of radioactive material

within the cell walls would obviously make itdifficult to interpret any experiments designed tofollow the transfer of radioactive material betweenfractions during the course of protein synthesis.This led us to turn our attention to studies on theprotoplast system, which had the twofold ad-vantage of eliminating the cell wall and reducingthe length of time required to fractionate thecellular components.

Experiment8 with protoplasts. In initial experi-ments, the great fragility of the protoplasta, com-bined with the necessity to maintain them understrictly aerobic conditions, made it difficult toobtain preparations that yielded consistent results.Conditions (described in the Methods section) wereeventually established that gave satisfactorilyreproducible results with our strain. When time-studies of the incorporation of labelled amino acidswere carried out, it was found that the specificradioactivities of membrane and cytoplasmicfractions showed much greater differences than inmany of the experiments carried out with wholecells. The membrane-protein fractions were againthose most highly labelled at early times, but, after5-7 min., the proteins of the cytoplasm attainedhigher specific radioactivities. Typical results withdifferent 14C-labelled amino acids are shown inFig. 3. The differences between the three sets ofcurves probably reflect differences in the degree ofdamage to the protoplasts and not differences inthe incorporation behaviour of the individualamino acids.

Experiments with broken protoplasts and i8olatedmembrane8. When protoplasts were disintegratedby rapid shaking in the Mickle tissue disintegratorand then incubated in the usual protoplast-in-corporation medium, it was found that "4C-labelledamino acids were taken up into the membraneprotein at rates comparable with those observed inintact protoplasts (Fig. 4). Very little radioactivitywas found in the cytoplasmic protein, however.This result was largely repeatable although in onesubsequent experiment the cytoplasmic proteinsreached a specific radioactivity of 25% of that ofthe membrane protein after incubation for 30 min.Much the same degree of incorporation of 14C-

labelled amino acids into membrane proteins wasfound if the membranes were separated from thecytoplasm by sedimentation and resuspended in'C' solution with 1 % added glucose before carryingout the incubation (Fig. 5). The presence of traces

of cytoplasm was not necessary because membranestwice washed with buffer were as active as mem-branes used immediately after sedimentation. Themethod used for breaking the protoplasts was alsounimportant, as it was found that membranes pre-pared from protoplasts lysed by diluting themedium three times with water incorporatedlabelled amino acids into protein just as well asthose obtained by breaking protoplasts in theMickle disintegrator. Finally, full activity of thesystem was retained if the protoplasts were lysedand the membranes collected and used afterstorage for 2 days at 20 (Table 2).The linear incorporation of labelled amino acids

into membrane protein was found to continue forat least 2 hr. with L-["4C]valine (Fig. 5). In otherexperiments, incubations were not continued forsuch a long period but the uptake was linear untilthe end of the experiment in all.

12 .

1.0o

0-8u

>1.0-6

._vla%n0

I* I0 10 20 30 40

Time (min.)Fig. 4. Incorporation of [l-14C]glycine into the protein of

the membrane fraction of disrupted proplasts suspendedat a density of 1 mg./ml. in a medium containing 0.5M-KH2PO4, other inorganic salts and glucose. The aminoacid (10 hc) was added to membranes derived from 1-5 g.(dry wt.) of whole cells. The disruption and subsequentanalysis are described in the Methods section.

Table 2. Incorporation of [1-14C]glycine into mem-branes prepared from lysed protoplasts stored at20 for varying lengths of timeIncorporation of 2,uc of labelled amino acid was carried

out for 30 mi.; 100 mg. of membranes was used in eachcase. Other experimental conditions were as described inthe text.

Time ofstorage of

lysed protoplasts(days)

0

12

Incorporation oflabelled amino

acids intomembrane protein

(Ic/g.)8

117

Vol. 69 549

J. A. V. BUTLER, A. R. CRATHORN AND G. D. HUNTERThe action of deoxyribonuclease on the system

was also examined. Membranes were incubatedwith the enzyme before and after incubation with14C-labelled amino acid and the results are sum-marized in Table 3. These treatments had littleeffect although in both a small amount of proteinwas solubilized. However, prolonged incubation of

2-2

bo, 1-4

1-2

cZ4)

0.4^1

0

the membranes at 370 with or without deoxyribo-nuclease did considerably reduce the rate at whichamino acids were incorporated into the protein ofthe membranes.

Transfer of labelled protein from membranes tocytopla&m. At the same time, repeated attemptswere made to obtain incorporation of "4C-labelledamino acids into the proteins of isolated cyto-plasm, diluted by the medium in which the proto-plasts were broken. A small incorporation wasobserved, never more than 10 % of the incorpora-tion into isolated membranes and usually muchless. The incorporation was not enhanced by in-cluding adenosine triphosphate, fructose 1:6-diphosphate or a complete mixture of amino acidsin the medium. However, it was found that a very.high degree of labelling of the cytoplasmic proteinscould be achieved if they were incubated withwashed membranes previously labelled in vitro asdescribed above. Further, the proportion of radio-active label that could be transferred from mem-brane to cytoplasm was dependent upon the timefor which the membranes had been incubated withthe [14C]amino acid. The results are summarized inTable 4, and it will be seen that no loss of labelled

Table 3. Effect of digestion with deoxyribonucleaseon the 8pecific radioactivity of the membrane proteinsMembranes (100 mg.) were treated at 370 for 1 hr. with

deoxyribonuclease (DNA-ase; 50 sg./ml.) in 10 ml. of 'C'solution-phosphate medium.The membranes were then sedimented and either re-

suspended for incubation or treated with trichloroaceticacid. Incubations were carried out with 2,uc of [1-14C]-glycine as described in the text.

Time (min.)Fig. 5. Incorporation of L-[14C]vahne into the proteins of

the membrane fraction isolated from disrupted proto-plasts of Bacilus megaterium. The amino acid (10jlc) wasadded to membranes derived from 1-5 g. of whole cellssuspended at 1 mg./ml. in a medium containing inorganicsalts and glucose. The preparation of the fraction andsubsequent analysis of the samples taken at varioustimes is described in the Methods section.

Treatment with DNA-asebefore incubating

membranes for 2 min. withlabelled amino acid

Specificactivityof protein

DNA-ase (1kc/g.)+ 0-36- 0-38

Treatment with DNA-aseafter incubating

membranes for 30 min. withlabelled amino acid

A.

Specificactivityof protein

DNA-ase (.Uc/g.)+ 3-9- 4-1

Table 4. Transfer of a labeUed protein fraction from membranes labelled in vitro with [1I_4C]glycineto the protein fraction of unlabelled cytoplam

In each case, the membranes (approx. dry wt. 42 mg.) were incubated with cytoplasm (approx. dry wt. 93 mg.) for5 min. All incubations were carried out aerobically at 300.

Time ofincubation of

membranes with[l-14C]glycine

(min.)1-5

1030

Initial totalradioactivityof membrane

proteins(#AmC)

2-72251

Radioactivityof membraneproteins after

incubation withcytoplasm(6mc)

1.11539

Finalradioactivity ofcytoplasmicproteins(Jtmc)

1-68

10

0-6

0-4

0-2

Transfer(%)593620

550 I958

SITE OF PROTEIN SYNTHESIS IN B. MEGATERIUM

material from the combined protein fractionsoccurs, and that the greatest percentage transferwas obtained with membranes that had beenallowed to incorporate for the shortest time.An attempt was made to determine the rate at

which labelled protein is transferred from themembrane to the cytoplasm, membranes whichhad been pre-incubated for 20 min. with [14C]_amino acid being used. It was concluded that thisis a very rapid process as the transfer was found tobe complete in 1 min., the smallest intervalmeasured. On the other hand, very little radio-activity (never more than 5 %) was lost from theprotein fraction of previously labelled membraneswhen they were incubated in buffer alone for30 min.

Analyses. The nucleic acid contents of wholecells, membranes and cytoplasm as determined bythe methods of Cerriotti (1952, 1955) are given inTable 5. The values obtained differ from those givenby Weibull (1953 b), but his preparative proceduresdiffered in that he prepared protoplasts in a sucrosemedium and, probably more important, subse-quently treated them with deoxyribonucleasebefore examining membranes and cytoplasm.

Crude nucleic acid isolated from the cytoplasmdirectly by precipitation of the proteins withsodium dodecyl sulphate was found by baseanalysis to consist of 89% of RNA and 11 % ofDNA, in good agreement with the results given bythe colour reactions. But the treatment of finelydivided membranes with sodium dodecyl sulphateliberated nucleic acid into solution that was shown,after purification, to contain only traces of RNA.Weibull's membranes contained very little RNA,but the bulk of the RNA could be extracted fromthe cytoplasm by centrifuging for 21 hr. at105 000 g and sedimentation studies showed thatthere were three macromolecular componentspresent with sedimentation coefficients of 42,26 and 3s. In our preparations sedimenta-tion analysis of the cytoplasm, performed byDr K. V. Shooter of this Institute, showed thatthere were three macromolecular components withsedimentation coefficients of 13, 6 and 3s andat least 80% of the material was present in the3 s component.

Table 5. Compossition of whole cells, cytoplmnicmembranes and cytoplasm of Bacillus megaterium,prepared as described in the text

Whole celLsCytoplasmic membranesCytoplasm

Dry wt. ofwhole cells

(%)1002943

DNA(%)

1-52-81-3

RNA(%)

8-03.79.9

The cytoplasm, as prepared by us, thus containsno particles comparable with microsomes. In factit can be estimated that the 3s component has amolecular weight of about 30 000.

DISCUSSION

The initial experiments with whole cells show thatalthough the cell wall contains large quantities offree amino acids, it plays very little part in, at least,the initial processes of protein synthesis in theremainder of the cell. It may act as a reservoir ofamino acids and provide a constant supply to theorganism. This could account for the consistency ofthe amount of unincorporated amino acids found inthe cytoplasm which are either in the free state orin the form of simple derivatives.In the whole-cell experiments the time required

for the digestion by lysozyme probably did resultin some equilibration between the labelled proteinof the membrane and cytoplasmic fractions,because, in the more rapidly fractionated protoplastexperiments, the differences in the labelling ofthese fractions are much more marked. Althoughall experiments show an initially greater labellingof the membrane, which is later exceeded by thecytoplasm, individual differences observed inexperiments are difficult to interpret. It is, how-ever, not easy to be certain that, in any oneexperiment, all the protoplasts are active or eventhat all of them are intact.However, the feature common to all these

curves strongly suggests that the initial incorpora-tion of amino acid into protein occurs in themembrane fraction and is followed by transfer tothe cytoplasm. The further rise, usually observedafter 6 min., in the specific activity of the proteinsof the cytoplasmic membranes is then explicable ifprotein, initially formed in the membranes, can beused, possibly after further modification forstructural or enzymic purposes in the membranesthemselves as well as in the cytoplasm.Our conclusions are supported by the results of

the experiments in which the isolated membraneswere labelled in vitro. It is to be noted that, inmany cases, a similar level of labelling was observedwith isolated membranes as with membranes fromwhole protoplasts. That the cytoplasm does notplay a vital part in this process is borne out by thefact that its complete removal by washing did notprevent incorporation. A suggestion that thecytoplasmic membranes of B. megaterium may be asite of protein synthesis has also been made else-where (McQuillen, 1956).The actual amount of amino acid incorporated

into the proteins is sufficient to show that themechanism studied cannot be dismissed as an in-significant process. Thus in one experiment an

Vol. 69 551

J. A. V. BUTLER, A. R. CRATHORN AND G. D. HUNTER

incorporation of 50 ,ug. of [ 1 -14C]glycine into 50 mg.of membrane proteins was observed; the labellingwas not reduced by dialysing the membranesagainst four changes of water at 10 before addingtrichloroacetic acid. This amount represents anincorporation equivalent to about 2 % of the pre-existing glycine residues.The length of time for which the membranes will

continue to incorporate 14C-labelled amino acidsinto their protein fraction is also remarkable, thesystem being much more active in this respect thanother systems in vitro already described. The partplayed by the cytoplasm in the intact cell is notclear. It appears to be the final repository of thebulk of the label and thus may be the eventualreceptacle of many newly formed proteins. Thetransfer of labelled protein from membranes tocytoplasm removed previously is a very rapidprocess and occurs without loss of labelled protein.This suggests that protein is being transferredrather than amino acids or simple derivatives and isin itself indirect confirmation that the processesstudied in vitro are closely allied to protein syn-thesis.Much less radioactivity appeared in the cyto'

plasmic-protein fraction in the broken-protoplastexperiments than in the transfer experiments. Thereason for this discrepancy is not clear but could beexplained by production of a soluble transferinhibitor in the cytoplasmic membranes which isexcreted into the medium in the whole-protoplastexperiments and is also kept from contact with thecytoplasm by an intervening sedimentation in thetransfer experiments.We can conclude that these experiments give an

indication that the main site of protein synthesis inB. megaterium is the cytoplasmic membrane. Itmust be remembered that the 'membrane fraction'obtained in these experiments includes not only theactual protoplast membrane, but also the lipidgranules and part, at any rate, of the DNA gel.However, the cytoplasmic membranes themselvesconstitute the bulk (75%) of the fraction, andthere seems little doubt that they provide the sitesfor the incorporation of labelled amino acids. Thusour experiments with deoxyribonuclease make itseem unlikely that DNA or protein associated withit plays an important role in the incorporationprocess. This is further borne out by the experi-ments with EDTA which showed, although theresults obtained were rather variable, that proteinsolubilized with the bulk of the DNA, and possiblyassociated with it, was not as readily labelled asthe residual protein. Besides, a large proportion ofthe cellular DNA occurs in the cytoplasmic frac-tion, and the material of the original DNA-con-taining gel is probably distributed almost equallybetween our membrane and cytoplasmic fractions.

It seems most unlikely that the lipid granules playany direct part in protein synthesis: these also aredigested by EDTA. The 'small-particle' fractionobtained by Weibull (1953b) is absent from ourcytoplasm. Whether this arises from the dissolutionof the gel by Weibull's deoxyribonuclease or thecytoplasmic membranes is an open question; butMitchell & Moyle (1956) have shown that the'small-particle' fraction of Staphylococcus aureu8probably arises as a result of the disintegration ofthe delicate cytoplasmic membrane of thatorganism. Methods offractionating the cytoplasmicmembranes are at present being studied in moredetail.

SUMMARY

1. The incorporation of 14C-labelled amino acidsinto protein has been studied in whole cells ofBacillw megaterium and also in protoplasts andvarious subcellular fractions derived from the sameorganism.

2. The cell wall does not incorporate any of theamino acids studied into its 'protein' to any greatextent, but it absorbs a large quantity of free aminoacid.

3. Initially, labelled amino acids are incorpor-ated most rapidly into the protein derived from thecytoplasmic membranes of whole cells and proto-plasts incubated in growth media. At later timesthe cytoplasmic proteins attain higher specificactivities. At all times the level of the non-proteinradioactivity is highest in the cytoplasm.

4. Isolated cytoplasmic membranes can in-corporate 14C-labelled amino acids into their proteinfractions at rates comparable with those observedin the membranes of whole protoplasts: the in-corporation is linear for at least 2 hr. In com-parison, isolated cytoplasm incorporated verysmall amounts of amino acid into protein.

5. If membranes labelled in this way are incu-bated with non-labelled cytoplasm, a considerableproportion of the radioactivity is rapidly trans-ferred from the membrane protein to the cyto-plasmic protein.

6. It is concluded that the initial stages ofprotein synthesis in B. megaterium take place atsites on or closely associated with the cytoplasmicmembrane.

The authors would like to thank Mr R. Goodsall andMrs J. Q. Tapley for invaluable technical assistance.The investigation has been supported by grants to the

Chester Beatty Research Institute (Institute of CancerResearch: Royal Cancer Hospital) from the British EmpireCancer Campaign, the Jane Coffin Childs Memorial Fundfor Medical Research, the Anna Fuller Fund and theNational Cancer Institute of the National Institutes ofHealth, U.S. Public Health Service.

552 1958

Vol. 69 SITE OF PROTEIN SYNTHESIS IN B. MEGATERIUM 553

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Cerriotti, G. (1952). J. biol. Chem. 198, 297.Cerriotti, G. (1955). J. biol. Chem. 214, 59.Cohn, P. & Butler, J. A. V. (1957). Biochim. biophys. Acta,

25, 222.Cowie, D. B. & Walton, B. P. (1956). Biochim. biophys.

Acta, 21, 211.Crathorn, A. R. & Hunter, G. D. (1957). Biochem. J. 67,37.Crathorn, A. R. & Hunter, G. D. (1958). Biochem. J. 68,

4P.Gale, E. F. & Folkes, J. F. (1955). Biochem. J. 59, 661.Hoagland, M. B., Keller, E. B. & Zamecnik, P. C. (1956).

J. biol. Chem. 218, 345.Hoagland, M. B., Zameenik, P. C. & Stephenson, M. L.

(1957). Biochim. biophys. Acta, 24, 215.Hunter, G. D. & Butler, J. A. V. (1956). Biochim. biophys.

Acta, 20, 405.

Hunter, G. D., Crathorn, A. R. & Butler, J. A. V. (1957).Nature, Lond., 180, 383.

Keller, E. B., Zamecnik, P. C. & Loftfield, R. B. (1954).J. Hi8tochem. Cytochem. 2, 378.

Littlefield, J. W., Keller, E. B., Gross, J. & Zamecnik, P. C.(1955). J. biol. Chem. 217, 111.

Marshak, A. & Vogel, H. J. (1951). J. biol. Chem. 189,597.

McQuillen, K. (1955). Biochim. biophy8. Acta, 17, 382.McQuillen, K. (1956). Bacterial Anatomy, p. 127. Cam-

bridge University Press.Mitchell, P. & Moyle, J. (1956). Bacterial Anatomy, p. 172.

Cambridge University Press.Popjak, G. (1950). Biochem. J. 46, 560.Simkin, J. L. & Work, T. S. (1957). Biochem. J. 65, 307.Vennes, J. W. & Gerhardt, P. (1956). Science, 124, 535.Weibull, C. (1953 a). J. Bact. 66, 688.Weibull, C. (1953 b). J. Bact. 66, 696.Weibull, C. (1955). Exp. Cell Res. 9, 139.Wyatt, G. R. (1951). Biochem. J. 48, 584.

The Metabolic Stability of the Nucleic Acids in Culturesof a Pure Strain of Mammalian Cells

BY R. Y. THOMSON, J. PAUL AND J. N. DAVIDSONDepartment of Hiochemi8try, The Univer8ity of Gla8gow

(Received 5 November 1957)

Although in recent years the metabolism of thenucleic acids has been the subject of many experi-ments with isotopic-tracer techniques, our know-ledge of the relative rates at which deoxyribonucleicacid (DNA), nuclear ribonucleic acid (nRNA) andcytoplasmic ribonucleic acid (cRNA) aresynthesizedand broken down is still far from complete.The present experiments were designed to

provide answers to two particular questions: (1)What inferences about nucleic acid synthesis canbe drawn from the manner in which [14C]formate isincorporated into the purines and pyrimidines ofDNA, nRNA and cRNA? (2) To what extent areDNA, nRNA and cRNA, once synthesized, meta-bolically stable in animal cells?

Information of this kind cannot readily be ob-tained from the intact animal or from tissue slicessurviving in vitro. Use was therefore made of apure strain of mammalian cells growing in tissueculture. Brief preliminary reports of some of theresults obtained have already been published(Thomson, Paul & Davidson, 1956; Thomson &Paul, 1957).

METHODS

BiologicalThe cells used in these experiments were adult mouse sub-cutaneous fibroblasts NCTC strain L, clone 929 (Sanford,Earle & Likely, 1948), hereafter referred to as L cells. These

cells were grown directly on the floor of large conicalculture flasks having an area ofabout 140 cm.2. The mediumin which they were normally maintained was composed of20 parts of horse serum, 10 parts of chick-embryo extractand 70 parts of Hanks's balanced salt solution (Hanks &Wallace, 1949). It also contained 50 units of sodiumpenicillin Gfml. but no other antibiotics.

In most of the experiments to be described, similarvessels were used. In each vessel was placed a glass-coveredmagnet so that the cells could readily be suspended byplacing the culture vessel on a magnetic stirrer. This pro-cedure proved less damaging to the cells than treatmentwith trypsin. In the conditions described the cells usuallyreached a maximum population of between 50 and 80millions/flask, at which level the population becamestationary. In all experiments the population was keptnear the maximum, the cultures being halved at eachtransfer and allowed to grow to the maximum once more.The medium, 60 ml./flask, was completely renewed twicea week and on one of these occasions the cells were trans-ferred.

In this way, a slow rate of growth was maintained andthe cells remained healthy throughout. In some experi-ments, where it was necessary to exclude extracellularpools of purines and pyrimidines, the medium used wasEagle's medium (Eagle, 1955) supplemented with 30%(v/v) of dialysed horse serum. Also, in some experimentswhere the inocula of cells were much smaller than thosedescribed above, smaller vessels were used, e.g. 25 ml.Erlenmeyer flasks.At the commencement of an experiment a suspension of

cells was obtained by treating stock cultures of cells with